Thermostable variants of neutral proteases of Bacillus stearothermophilus and Bacillus thermoproteolyticus

ABSTRACT

The present invention provides genes encoding extremely thermostable variants of neutral proteases, as well as, vectors and cells comprising these genes and proteases produced through these genes, vectors and/or cells. In particular variants of the neutral proteases of Bacillus stearothermophilus and Bacillus thermoproteolyticus (thermolysine) and sequences and cells coding therefor are provided.

This is a U.S. national stage application of PCT/NL95/00037 filed Jan.26, 1995.

The present invention relates to metallo-endopeptidases, also calledneutral proteases (NP's), that are produced, processed and secreted bymembers of the bacterial genus Bacillus. More specifically, it relatesto the use of genetic manipulation techniques to alter genes encodingneutral proteases, such that the thermostability of the enzymes encodedby these genes is increased. The altered genes are also a part of thepresent invention, as are vectors and cells comprising said genes. Theinvention also relates to the production, processing and secretion ofmore stable neutral proteases derived from B. stearothermophilus by a B.subtilis strain carrying genes encoding these enzymes. Morespecifically, this invention relates to the introduction of a series ofspecific site-directed mutations in the gene encoding the neutralprotease of B. stearothermophilus, which increase the thermostability ofthe product encoded by this gene. It also relates to a general analysisof the structural elements that determine the thermostability of theneutral protease of B. stearothermophilus and to the application of theresults of this analysis for the stabilization of other neutralproteases, in particular thermolysin. The altered genes coding for themore stable thermolysin, as well as cells and vectors comprising suchgenes are also disclosed in the present invention. The invention alsorelates to the products of such genes, vectors and cells.

A general problem in the industrial use of enzymes is the stability ofthese catalysts. Enzymes are expensive and should preferably be usablein as low quantities and in as many process cycles as feasible. It isoften desirable to conduct industrial processes at elevated temperatures(Nosoh and Sekiguchi, 1990; Kristjansson, 1989; Geisow, 1991). Thislimits the use of enzymes, since many enzymes do not sufficientlytolerate temperatures outside the physiological range. Thermostableenzymes are often more stable in general (Sonnleitner and Fiechter,1983; Nosoh and Sekiguchi, 1990): they are more stable at anytemperature and they have a higher resistance towards other denaturingfactors such as extreme pH values, detergents and high saltconcentrations. The availability of thermostable enzymes is thereforeclearly desirable for the following reasons:

The temperature range at which enzymatically catalyzed processes can beconducted is expanded.

The enzymes last longer thus reducing the quantities needed.

NP's are used in several industrial processes, the most important ofwhich is the preparation of the artificial sweetener aspartame(Gerhartz, 1990; Isowa et al., 1979). NP's are also employed in theleather and baking industry, in breweries, and in the production ofprotein hydrolysates in the leather industry and in breweries (Gerhartz,1990). At present, mostly thermolysin (e.g. in the preparation ofaspartame) and the neutral protease of Bacillus subtilis (e.g. forbeer-brewing applications) are used in industrial processes. The costsof these processes could be reduced if more stable NP variants would beavailable. A recent paper by Kubo et al. (1992) is an illustration ofthe interest of the aspartame industry in this matter.

Several Bacilli are known to produce extra-cellularmetallo-endopeptidases, also called neutral proteases. These enzymescontain 300-319 residues and are active in the neutral pH range. Thebest known NP is thermolysin, the highly thermostable 316 residue NPfrom Bacillus thermoproteolyticus. Bacilli exhibit considerabledifferences in growth temperature and the thermostabilities of theirneutral proteases differ accordingly. Several Bacillus neutral proteaseshave been characterized and genes encoding for these enzymes have beencloned and sequenced from e.g. B. subtilis (Yang et al., 1984), B.stearothermophilus CU-21 (Fujii et al., 1983; Takagi et al., 1985), B.stearothermophilus MK-232 (Kubo & Imanaka, 1988), B. thermoproteolyticusrokko (Marquardt et al., 1990) and B. caldolyticus (Van den Burg et al.,1991). Using genetical techniques (such as `site-directed mutagenesis`,e.g. Stanssens et al., 1989) genes encoding neutral proteases have beenmutated to change properties of the enzymes, such as thermostability(Imanaka et al., 1986; Toma et al., 1991; Van den Burg, 1991; Eijsink,1991).

A piece of chromosomal B. stearothermophilus DNA containing the neutralprotease gene (npr) has been cloned by Fujii et al. (1983) into plasmidpTB90; the resulting plasmid was designated pNP22. The npr gene wassubcloned from pNP22 into pTZ12 (Aoki et al., 1987), resulting in pGE501(Eijsink et al., 1990, 1992b). Plasmid pGE501 could be transformed toBacillus subtilis strain DB 117 (Eijsink et al., 1990). Bacillussubtilis DB117 cells containing pGE501 (or a derivative thereof; seebelow) expressed, processed and secreted the neutral protease B.stearothermophilus (NP-ste). From plasmid pGE501 fragments containingparts of the neutral protease gene have been subcloned into E. coliplasmids pMa and pMc for site-directed mutagenesis according toStanssens et al. (1989) (Eijsink et al., 1990). After site-directedmutagenesis npr genes containing the mutated fragment werereconstructed, yielding variants of pGE501. These variants of pGE501were essentially identical to the original pGE501 plasmid, with theexception of one or more nucleotide changes in the npr gene that gaverise to one or more amino acid changes in the mature gene product. Thisgenetic strategy has been used extensively by the inventors and severalsite-directed mutations in the npr gene that result in increasedthermostability of the gene product have been described (Eijsink, 1991;Eijsink et al., 1991a; Van den Burg et al., 1991; Vriend et al., 1991;Eijsink et al., 1992a,b; Hardy et al., 1993; Eijsink et al., 1993).

The present invention povides strategies for the construction ofextremely thermostable neutral proteases, which are far more stable thanany naturally occurring or engineered neutral protease described so far.

It also provides the products resulting from said strategies as well asthe use of said products in industrial processes and intermediates inmaking the products.

In the description of the present invention the thermostability of aneutral protease is defined by the `T50` of the enzyme. The T50 isdetermined as follows: The B. stearothermophilus neutral protease isproduced by a B. subtilis strain harbouring pGE501 or a mutated variantthereof and purified from the culture medium according to previouslydescribed methods (Van den Burg et al., 1989; Eijsink et al., 1991b).The purified enzyme is diluted to ±0.1 μM in 20 mM NaAc, pH 5.3, 5 mMCaCl₂, 0.5% isopropanol and 62.5 mM NaCl (final concentrations), andincubated for 30 minutes at various temperatures, ranging from 58 to 98°C. After incubation the residual proteolytic activity is determined andexpressed as percentage of the initial activity. T50 is the temperatureat which 50% of the initial activity is retained after the 30 minutesincubation period. In every thermostability assay a reference samplecontaining the wild-type B. stearothermophilus neutral protease isincluded and the thermostability of mutant neutral proteases isexpressed as dT50, being the difference in T50 between the mutant andthe wild-type enzyme. Temperatures were registered using mercurythermometers. Since in earlier publications results obtained usingalcohol thermometers were given, and since the pH in the presentthermostability assay was slightly higher than in earlier assays, thepresent data deviate slightly from earlier published data. Deviations indT50 values are small. Deviations in T50 values are considerable (causedby the use of mercury instead of alcohol thermometers) and amount uptoplus 6° C. in the >90° C. temperature range.

On the basis of previous results on NP thermostability it has beenspeculated that, at least under the conditions of the thermostabilityassay used in the present invention, the thermostability of an NP, andthus its T50, is determined by the rate at which local unfoldingprocesses, that render the NP molecule susceptible to autolysis, occur(Eijsink et al., 1991c, 1992c; Vriend and Eijsink, 1993; see alsoDahlquist et al., 1976 and Braxton and Wells, 1992). This presumedmechanism has several consequences (Vriend and Eijsink, 1993):

Mutations with large effects on thermostability of NPs should be mainlylocated at the surface of the protein. This was indeed observed inNP-ste (Eijsink et al., 1992b; Vriend and Eijsink, 1993).

Mutations with large positive or negative effects on NP thermostabilityshould be located in regions that unfold relatively easily upon heating.Mutations aimed at stabilizing NPs are most effective if they areintroduced in a region that unfolds relatively easily (in the remainderof the description such a region is called a `weak region`). For NP-stethis consequence was partly sustained by experimental evidence: manymutations, in several parts of the protein had only marginal effects onthermostability, whereas mutations in the 66-72 region had large effectson thermostability. This suggests that the 66-72 region is part of aweak region in the enzyme (Vriend and Eijsink, 1993).

The identification of weak regions is essential for the successfuldesign of a mutation strategy to stabilize NPs. In addition it isessential to know the relative importance and the total number of weakregions, as explained by Eijsink et al. (1992c).

The effects of mutations that stabilize the same local region of the NP(or, in other words, that affect the same pathway of local unfolding)are not additive. It is no use to `over-stabilize` a weak region,because, after sufficient stabilization, unfolding in this region willno longer play a role in the overall process of thermal inactivation.This was indeed observed (Eijsink et al., 1992c). As long as theaddition of extra stabilizing mutations to a region of the proteinresults in increased thermostability, this region must be considered asrelatively weak. It means that any following stabilizing mutation inthat region will increase the thermostability of the enzyme. Thus thatregion should be considered during the design of further stabilizingmutations. This is described in the present invention. In general, it isnot to be expected that many mutations in the same region of the proteinexhibit considerable additivity. This is only possible in case an NP hasone weak region which is considerably weaker than any other unfoldingregion. Such a case is described in the present invention.

Information derived from a comparison of naturally occurringthermostable and thermolabile neutral proteases could help in the designof stabilization strategies for NPs. However, despite the availabilityof the primary structures of many NPs and the tertiary structures of anextremely thermostable (thermolysin; Holmes and Matthews, 1982) and aless thermostable (from Bacillus cereus; Stark et al., 1992) NP, thestructural factors underlying the differences in thermostability betweennaturally occurring neutral proteases are largely unknown (see e.g.Stark et al., 1992). Site-directed mutagenesis studies have providedsome insight in the structural differences that underly the differencein thermostability between thermolysin (T50=86.9° C.) and the lessstable NP-ste (T50=73.4° C.; NP-ste and thermolysin have 85% sequenceidentity). This is shown in Table I.

Table I. Previously published analyses of the contribution of sequencedifferences between NP-ste and thermolysin to the difference inthermostability between these two enzymes. Residues in NP-ste (T50=73.4°C.) were replaced by the corresponding residue in thermolysin (T50=86.9°C.).

    ______________________________________                                                     dT50                                                                                      Mutation: (                                                                  ° C.) .sup.1) : Reference:                     ______________________________________                                        Ala4 -> Thr  +1.9       Van den Burg et al., 1991                               Gly47-Arg48-Asn49 -> -0.2 Eijsink et al., 1993                                Ala-Lys-Tyr                                                                   Thr59 -> Ala +1.9 Van den Burg et al., 1991                                   Gly61 -> Ala .sup.2) Imanaka et al., 1986                                     Thr66 -> Phe +7.0 Van den Burg et al., 1991                                   Ala72 -> Pro +6.3 Hardy et al., 1993                                          Gly92 -> Asn -0.3 Eijsink et al., 1993                                        Gly144 -> Ala +0.6 Imanaka et al., 1986;                                        Eijsink et al., 1993                                                        Arg185 -> Lys -2.7 Eijsink et al., 1993                                       Ala204 -> Ser -0.4 Eijsink et al., 1993                                       Cys291 -> Ala 0 Eijsink et al., 1992b, 1993                                   Asn314 -> Asp -0.3 Eijsink et al., 1993                                     ______________________________________                                         .sup.1) Some dT50 values differ slightly from earlier published values,       for reasons described above.                                                  .sup.2) This mutation was mentioned in a publication by Imanaka et al.        (1986). In that publication stability data for this mutant are not given.     The T.sub.50 value for this mutation has not been published.             

Since NP-ste and thermolysin have 85 percent sequence identity, theseenzymes are expected to be highly similiar with respect to overallstructure. Statistical analyses of similarities between the structuresof homologous proteins have provided compelling evidence supporting thisassumption (Chothia and Lesk, 1986; Sander and Schneider, 1991; Vriendand Eijsink, 1993). The high similarity between the two enzymes makes itplausible that the rate of their thermal inactivation is determined byunfolding of the same regions. The relative contribution of unfolding ofthe different weak regions to the overall thermal inactivation rate mustdiffer between the two enzymes, since they have differentthermostabilities. Because of the high similarity between the twoenzymes it is also plausible that the introduction of mutations inthermolysin, that are reciprocal to the ones listed in Table 1, haveeffects on the thermostability of thermolysin that are reciprocal to theeffects listed in Table I (at least qualitatively).

From the data in Table I and from results described by Eijsink et al.(1992b) it was concluded that the 66-72 region of NP-ste might be aregion that unfolds relatively easily. In accordance with thisconclusion and its assumed consequences (see above), it appeared thatmutations at two other positions in this region (65 and 66) hadrelatively large effects on thermostability (Hardy et al., 1993).Replacing Ser65 by Pro increased the T50 of NP-ste by 4.7° C. (Hardy etal., 1993). The latter mutation was a so-called `designed` mutation. Inthe present invention mutations are called `designed` in case they havenot been suggested by nature (through comparisons of homologous NPvariants); instead, they have been `designed` on the basis of theprinciples that (are generally supposed to) govern protein structure andstability.

On the basis of the results and theory described above, the aim ofstabilizing NP-ste seems to be attainable by the following steps:

Combine the stabilizing mutations listed in Table I.

Analyze the importance of other structural differences between NP-steand thermolysin. As a result of this analysis, essential knowledgeconcerning the structural factors that determine NP thermostability maybe obtained. This knowledge is needed for the design of furtherstabilizing mutations. In addition, this analysis may result in theidentification of `new` mutations in NP-ste, that have a stabilizingeffect. Thirdly, this analysis may help in identifying weak regions inthe NPs.

Design and insert stabilizing mutations that stabilize against localunfolding in the 66-72 weak region of NP-ste.

Identify other weak regions (that is regions in which mutations haverelatively large effects on thermostability) and introduce stabilizingmutations in those regions.

Combine all identified stabilizing mutations in all identified weakregions. Per weak region, the addition of stabilizing mutations shouldbe continued till no further additivity is observed.

The novel strategy provided in the present invention gives a vast arrayof possibilities for obtaining additional mutated neutral proteases withenhanced thermostability.

In addition the invention provides unexpectedly stable proteases, whichare more stable than thermolysin itself. Surprisingly, by providing morethan one mutation in a weak region, optionally in combination withmutations in other weak regions, the thermostability still increased.

Moreover, it was unexpected that combinations of mutations designed onthe basis of thermolysin would lead to more thermostable proteases thanthermolysin itself.

From the foregoing it may be clear for what purpose the novel mutatedproteases according to the invention can be used. Additionalapplications such as preparation of protein hydrolysates, etc. arereadily available for the person skilled in the art.

The person skilled in the art will also realize that it is possible touse active fragments or derivatives of the proteases disclosed in thepresent invention. These fragments need only to be active andthermostable. Derivatives can be produced by changing amino acidresidues in regions where they do not influence activity and/orthermostability.

The present invention provides essential information that enables evenfurther stabilization of NP-ste through site-directed mutations. Thepresent invention also shows clearly how thermolysin could be stabilizedthrough site-directed mutations. Apart form the mutations in NP-ste andthermolysin that are obvious from the present invention, one could thinkof:

Rigidification of the N-terminal domain, especially of the 59-72 regionand the N-terminal beta-hairpin (residues 1-25), by introduction ofproline residues or by the introduction of disulfide bonds.

Mutations in the weak regions of NP-ste and thermolysin (as describedand/or identified in the present invention), that are designed on thebasis of general principles regarding protein structure and stability.

Identification of the auto-digestion sites that play a rate-limitingrole in the process of thermal inactivation. Alteration of theseautodigestion sites by site-directed mutations.

In relation to the use of enzymes in industry, often attempts to improveand change the specificity and activity of enzymes are made, usingsite-directed mutagenesis techniques. The enzymes in the presentinvention are a good starting point for such attempts. Successfulmanipulation of activity and specificity of the extremely stable enzymespresented here, would result in enzymes that are even more valuable forindustry.

The extremely stable enzymes presented here are expected to be stableunder several harsh conditions, since thermostable enzymes are oftenalso relatively stable under for example extremes of pH or high saltconcentrations (Sonnleitner and Fiechter, 1983). This increases thepotential use of these enzymes in industry.

For reference, the experimental results related to the present inventioncan be compared to the results obtained by Imanaka et al. (1986), whodescribed two stabilizing site-directed mutations in the neutralprotease of B. stearothermophilus. They used an assay forthermostability that differs from the one used in the description of thepresent invention. The stabilizing replacement of Gly144 by Aladescribed by Imanaka et al. resulted in a dT50 of +0.6° C. in thethermostability assay used in relation to the present invention. Thedestabilizing replacement of Thr66 by Ser described by Imanaka et al.resulted in a dT50 of -4.4° C. in the thermostability assay used inrelation to the present invention.

Methods

Plasmids and Strains

The B. stearothermophilus npr gene (taken from plasmid pNP22; Takagi etal., 1985; Fujii et al., 1983) was subcloned in the high copy number B.subtilis vector pTZ12 (Aoki et al., 1987, yieding pGE501 (Eijsink etal., 1990, 1992b). The gene was expressed in the protease deficient B.subtilis strain DB117 (Eijsink et al., 1990). Suitable fragments of thenpr genes were subcloned in the E. coli plasmid pMa/c for site-directedmutagenesis (Stanssens et al., 1989). E. coli WK6 and WK6MutS (Zell andFritz, 1987) were used in site-directed mutagenesis procedures. Allstrains were grown on Trypton-Yeast medium, containing appropriateantibiotics.

Site-directed Mutagenesis

The design of the mutagenic oligonucleotides used for the production ofsite specific mutations was such that a restriction site was removed orcreated to facilitate the selection of mutants, without producingadditional changes at the amino acid level (Eijsink et al., 1990;Eijsink et al., 1992 c). Mutagenesis was performed using a gapped duplexmethod described earlier (Stanssens et al., 1989). Mutant clones wereselected by restriction analysis and their npr gene fragment wassequenced using the dideoxy chain termination method (Sanger et al.,1977). For the production of mutant neutral proteases in B. subtilis,correctly mutated npr gene fragments were used to construct derivativesof pGE501 containing an intact mutant npr gene. Multiple mutations wereconstructed either by performing several subsequent mutagenesis steps(each step followed by sequence analysis of the mutated gene fragment),or by combining fragments of mutant genes using standard genetictechniques (Sambrook et al., 1989).

Production, Purification and Characterization of Neutral Proteases

B. subtilis DB117 harbouring a pGE501 variant was cultured in 600 mlmedium in aerated 1000 ml flasks at 32° C. After 16 hours of cultivationthe cells were removed by centrifugation and the supernatants wereloaded onto Bacitracin-silica columns (Unilever Research Laboratories,Vlaardingen, The Netherlands) for affinity chromatography as describedbefore (Van den Burg et al., 1989). After purification the enzymes werestored at -18° C. in the elution buffer used in the affinitychromatography procedure (20 mM sodium acetate, pH 5.3; 5 mM CaCl₂, 20%(v/v) isopropanol; 2.5 M NaCl; 0.03% sodium azide). Purified enzyme wasanalysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) as described before (Van den Burg et al., 1989).

For the determination of T50, aliquots of diluted pure enzyme(approximately 0.1 μM in 20 mM sodium acetate, pH 5.3, 5 mM CaCl₂, 0.5%(v/v) isopropanol, 62.5 mM NaCl) were incubated at appropriatetemperatures. Subsequently, the residual protease activity wasdetermined using a caseine assay (Fujii et al., 1983). The T50 and dT50values presented in this invention are average values derived from atleast three independent measurements. The standard deviation in T50values is approximately 0.7° C.; the standard deviation in dT50 valuesis approximately 0.4° C.

The thermostability of NPs in time was determined under the sameconditions as those used in the T50 determination. The enzyme wasdiluted and divided into aliquots. Some of the aliquots were stored onice, whereas the others were incubated at 90° C. Aliquots were removedfrom the 90° C. incubator after different time intervals andsubsequently stored on ice. At the end of the incubation period the(remaining) proteolytic activity in all aliquots was determined usingthe casein assay.

The reference thermolysin sample was purchased from Boehringer Mannheim.Before usage the enzyme was purified in a way identical to that employedfor NP-ste.

Protein concentrations were determined using the MicroBCA assay suppliedby Pierce.

Design and Structural Analysis of Mutations

The design of stabilizing mutations, theoretical analysis of thestructural effects of mutations, and structural inspections of NPmolecules were performed using in computo procedures that haveextensively been described elsewhere (Eijsink et al., 1990; Vriend andEijsink, 1993). The design of stabilizing mutations and all theoreticalconsiderations were based on generally accepted principles of proteinstructure and stability as described by for example Matthews (1991),Alber (1991), Vriend and Eijsink (1993), Fersht and Serrano (1993).

Alternative Methods

In the present invention mutations were introduced by gapped-duplexsite-directed mutagenesis into the gene encoding NP-ste. There areseveral alternative methods to obtain the variants of the NP-ste genethat are equivalent to the ones described in the present invention:

The use of other techniques for site-directed mutagenesis, such as PCRbased methods.

The exchange of (part of) the natural gene by a synthetic gene thatcontains the correct mutations.

Mutagenesis of genes encoding other NPs (for example thermolysin), suchas to obtain genes that are identical to the ones described in thepresent invention. There are also several methods to obtain genes thatare not identical to the ones described in the present invention, butthat encode neutral proteases that are, with respect to structure andthermostability, essentially identical to the gene products described inthe present invention:

Construct NP-ste variants that contain the stabilizing mutationsdescribed in the present invention plus one or more mutations that donot dramatically affect thermostability.

Construct variants of thermolysin in which residues have been replacedby the corresponding residue in NP-ste, for example Asn19→Tyr, Ser103(106)→Thr en Lys182 (185)→Arg. In addition Ser68 may be replaced by Aspor Pro.

Construct chimeric (maybe partly synthetic) genes encoding NPs thatcontain the N-terminal part of thermolysin (Kubo et al., 1988; Marquardtet al., 1990) and the C-terminal part of wild-type NP-ste. The borderbetween the thermolysin part and the NP-ste part could for example liebetween residues 70 and 100.

The use of partly or completely synthetic genes encoding neutralproteases that, with respect to thermostability, are essentiallyidentical to the ones described above.

A more general alternative method that is based on the present inventionand that would result in genes encoding neutral proteases that, withrespect to thermostability, are essentially identical to the onesdescribed above is the following:

Introduce stabilizing mutations in the weak regions that have beenidentified in the present invention. NP-ste and thermolysin are likelyto have the same weak regions, so this strategy could be conducted usingeither thermolysin or NP-ste as a starting point.

Alignment of Neutral Protease Amino Acid Sequences

NP-ste contains 319 residues (Takagi et al., 1985), see FIG. 1 andthermolysin contains 316 residues (Titani et al., 1972; Marquardt etal., 1990), see FIG. 2. In the present invention, the sequences of thesetwo enzymes are aligned as depicted in FIG. 3. Residues `correspond` ifthey are at the same position in the alignment depicted in FIG. 3. As aconsequence of the extra three residues in NP-ste in the 25-30 region,the numbering of residues differs slightly between NP-ste andthermolysin. Some examples: Ala 4 in NP-ste (abbreviated to A in FIG. 3)corresponds with Thr 4 in thermolysin (abbreviated to T in FIG. 3); Arg185 in NP-ste (abbreviated to R in FIG. 3) corresponds to Lys 182(abbreviated to K in FIG. 3) in thermolysin.

Analysis of Differences Between NP-ste and Thermolysin

The gene encoding NP-ste was manipulated such as to change residues inthe mature gene product by residues that occur at the correspondingposition in thermolysin. The thermostabilities of the various mutantsare listed in Table II.

Table II. Analyses of the contribution of sequence differences betweenNP-ste and thermolysin to the difference in thermostability betweenthese two enzymes. Residues in NP-ste were replaced by the correspondingresidue in thermolysin.

    ______________________________________                                        Mutation       dT50 (° C.)                                             ______________________________________                                        Tyr19 -> Asn   -2.7                                                             Gly61 -> Ala +3.9                                                             Thr103 -> Ser -5.9                                                          ______________________________________                                    

Designed Stabilizing Mutations in NP-ste

The gene encoding NP-ste was manipulated such as to change Ser68 in themature gene product by Asp. This resulted in an increase of T50 by 3.2°C. This mutation was designed on the basis of general principlesgoverning protein structure and stability.

Construction of Extremely Stable Variants of NP-ste

The gene encoding NP-ste was manipulated such as to change severalresidues in the mature gene product (as compared to the NP-ste wild-typeenzyme). This resulted in changes in T50, as listed in Table III anddepicted in FIGS. 4 and 5. Table III. Manipulation of the gene encodingNP-ste, such as to change several residues in the mature gene product;T50 of the mutant enzymes and the effect of the mutations (dT50, ascompared to wild-type NP-ste) are given. For reference, thethermostabilities of thermolysin and NP-ste are also indicated. Themutations are indicated by number only; they concern Ala4→Thr (4),Thr59→Ala (59), Gly61→Ala (61), Thr66→Phe (66), Ser68→Asp (68-I),Ser68→Pro (68-II), Ala72→Pro (72)

    ______________________________________                                        Mutations          dT50 (° C.)                                                                      T50 (° C.)                                ______________________________________                                        NP-ste             0         73.4                                               thermolysin +13.5 86.9                                                        66 + 72 +12.3 85.7                                                            59 + 66 + 72 +14.4 87.8                                                       61 + 66 + 72 +15.7 89.1                                                       59 + 61 + 66 + 72 +18.8 92.2                                                   4 + 59 + 66 + 72 +15.7 89.1                                                   4 + 61 + 66 + 72 +17.2 90.6                                                   4 + 59 + 61 + 66 + 72 +19.9 93.3                                             59 + 61 + 66 + 68-I + 72 +21.0 94.4                                           59 + 61 + 66 + 68-II + 72 +21.7 95.1                                           4 + 59 + 61 + 66 + 68-I + 72 +22.9 96.3                                       4 + 59 + 61 + 66 + 68-II + 72 +23.5 96.9                                   ______________________________________                                    

General Characterization of the Mutant Enzymes

All mutant enzymes were characterized (in purified form) by SDS-PAGE andby determination of their specific activity towards casein. Using theseanalyses, all mutant enzymes described in the present invention weresimilar to the wild-type NP-ste enzyme with respect to thesecharacteristics.

Identification and Characterization of Weak Regions in NP-ste

Large effects on thermostability indicate weak regions in the protein.The present results show that weak regions in NP-ste are located in the59-72 region, and around residues 4, 19, 106, and 185. The effects ofthe mutations in the different regions indicate the relativecontribution of each of the regions to the overall process of thermalinactivation.

NP-ste has a clearly weak region that can broadly be defined as theN-terminal half of the protein, or, more narrowly, as the region 59 till72. Unfolding in this region must be highly predominant during heatingof the protein, since mutations in the 59-72 result in extremestabilization of the enzyme. Apparently, the region is not maximallystabilized in the invented mutants, since, in general, additivity ofmutational effects is observed. Even the `last` additional mutationsstill increased thermostability (see Table III): Adding Ser68→Asp orSer68→Pro to the 4-59-61-66-72 five-fold mutant gave increases in T50 of3.0° C. and 3.6° C., respectively. This indicates that furtherstabilizations could be achieved by additional mutations in the 59-72region.

The residues 4, 19 and 106 are located relatively close to the 59-72stretch, whereas residue 185 is located relatively far away. It is notclear whether residues 4, 19 and 106 should be considered to be part ofthe same weak region as the 59-72 stretch. In other words, it is notclear whether mutations at positions 4, 19 and 106 affect the same localunfolding pathway as mutations in the 59-72 stretch. Adding Ala4→Thr tothe 59-61-66-68(I)-72 five-fold mutant or the 59-61-66-68(II)-72five-fold mutant gives increases in T50 of 1.9° C. and 1.8° C.,respectively. There are two possible explanations:

1. Residue 4 plays a role in a different local unfolding pathway.

2. Mutations at position 4 affect the same local unfolding pathway asmutations in the 59-72 stretch. Since this stretch is not yet maximallystabilized (see above), additivity of mutational effects is observed.

Regardless of the precise character of the local unfolding pathways inNP-ste, the present results and the theoretical considerations describedabove clearly indicate that stabilizing mutations at positions 4, 19,106 and 185 and in their environment might be useful for furtherstabilization of the protease.

Thermolysin and NP-ste are highly similar enzymes and therefore theconclusions concerning weak regions and stabilization strategies drawnon the basis of experiments with NP-ste also apply to a certain extentto thermolysin. The replacement of only three residues in the N-terminaldomain of NP-ste by the corresponding residue in thermolysin(combinations 59-66-72 and 61-66-72) was sufficient to make NP-ste morethermostable than thermolysin. Also, by replacing four or five residuesin the N-terminal domain of NP-ste by the corresponding residue inthermolysin, NP-ste variants were obtained that are extremely stable andconsiderably more thermostable than thermolysin. From theseobservations, the following conclusions with respect to thermolysin canbe drawn:

Thermolysin is not optimized for thermostability.

The N-terminal domain, especially the 56-69 (59-72) region is alsoimportant for the thermostability of thermolysin.

Site-directed mutations aimed at stabilizing thermolysin should involvethe regions around residues 4, 19, 56-69 (59-72), 103 (106), and 182(185).

Thermolysin can be stabilized by replacing residues by the correspondingresidue in NP-ste. This obviously does not apply to residues 4, 56 (59),58 (61), 63 (66) and 69 (72); it does apply to for example residues 19,103 (106), and 182 (185) and to other, so far unidentified residues.

These conclusions make clear how thermolysin could be stabilized bysite-directed mutations.

FIGURE LEGENDS

FIG. 1

Amino acid sequence of the mature extracellular neutral proteasesecreted by B. stearothemophilus CU-21 and nucleotide sequence of thecorresponding part of the nprT gene encoding this protein (Takagi etal., 1985). The amino acid sequence denoted by `NP-ste` in this figurebelongs to the protein that is referred to in this invention as `theneutral protease of B. stearothemophilus`. In the present invention thisprotein was obtained by expressing the nprT gene in B. subtilis. Thesequence that was published for this protein contained an Asn atposition 259 (Takagi et., 1985). Further sequence analysis showed thatthis was an error and that position 259 is a Thr as indicated in thefigure (unpublished observations by the inventors). The numbers in thefigure refer to the numbering of the amino acid sequence of the mature,secreted neutral protease.

FIG. 2

Amino acid sequence of the mature extracellular neutral proteasesecreted by B. thermoproteolyticus (Thermolysin; TLN) and nucleotidesequence of the corresponding part of the tin gene encoding this protein(Takagi et al., 1985). The amino acid sequence denoted by TLN in thisfigure differs at two positions (indicated by ***) from the sequenceoriginally described for Thermolysin (see Titani et al., 1972): it hasAsn at position 37 instead of Asp, and Gln at position 119 instead ofGlu. It is most likely that the sequence presented in this figure (whichwas derived from a nucleotide sequence) is more accurate than thesequence originally published by Titani et al. (1972) (which wasdetermined by direct sequencing of the protein). In this invention it isassumed that Thermolysin contains indeed Asn at position 37 and Gln atposition 119. The numbers in the figure refer to the numbering of theamino acid sequence of the mature, secreted neutral protease.

FIGS. 3A and 3B

Alignment of the amino acid sequences of the mature extracellularneutral proteases secreted by B. thermoproteolyticus (thermolysin, TLN;Marquardt et al., 1990; the corresponding part of the gene encoding thisprotease, tln, is indicated), and by B. stearothermophilus CU21 (NP-ste;Takagi et al., 1985; the corresponding part of the gene encoding thisprotease, nprT, is indicated).

The amino acid sequence denoted by `NP-ste` in this figure belongs tothe protein that is referred to in this invention as `the neutralprotease of B. stearothermophilus`. In the present invention thisprotein was obtained by expressing the nprT gene in B. subtilis. Thesequence that was published for this protein contained an Asn atposition 259 (Takagi et al., 1985). Further sequence analysis showedthat this was an error and that position 259 is a Thr as indicated inthe figure (unpublished observations by the inventors).

The amino acid sequence denoted by TLN in this figure differs at twopositions (indicated by ***) from the sequence originally described forthermolysin (see Titani et al., 1972): it has Asn at position 37 insteadof Asp, and Gln at position 119 instead of Glu. It is most likely thatthe sequence presented in this figure (which was derived from anucleotide sequence) is more accurate than the sequence originallypublished by Titani et al. (1972) (which was determined by directsequencing of the protein). In this invention it is assumed thatthermolysin contains indeed Asn at position 37 and Gln at position 119.Thermolysin was obtained form Boehringer Mannheim.

The numbers in the figure refer to the numbering of the amino acidsequences of the mature, secreted neutral proteases. Residues in NP-stethat differ from the corresponding residue in thermolysin are printed inbold letter type. Counting the three amino acid insertion in NP-ste asone difference, the total number of positions differing between NP-steand TLN is 44.

FIG. 4

Typical thermostability (T50) curves for the wild-type B.stearothermophilus neutral protease and two variants of this enzyme inwhich various amino acid residues were changed. The figure shows T50curves for wild-type NP-ste (+), thermolysin (Δ), and a five-fold (o;Thr4, Ala59, Ala61, Phe66 and Pro72) and a six-fold (+; Thr4, Ala59,Ala61, Phe66, Pro68 and Pro72) mutant of NP-ste. The thermostabilityassays were performed with enzymes that were purified from culturesupernatants of B. subtilis DB117 strains harbouring plasmid pGE501 or amutated variant thereof that carried respectively a wild-type or avariant of the gene encoding the B. stearothermophilus neutral protease.The picture shows the relative residual proteolytic activity in samplesthat were incubated for 30 minutes at various temperatures (pH=5.3).

FIG. 5

Stability of the wild-type B. stearothermophilus neutral protease (+),thermolysin (Δ) and the most stable neutral protease variant claimedhere (o), in time, at 90° C. The picture shows the relative residualproteolytic activity in samples that were incubated at 90° C., atpH=5.3, for different periods of time.

LITERATURE

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Braxton, S., & Wells, J. A. (1992). Incorporation of a stabilizing Ca²⁺-binding loop into subtilisin BPN'. Biochem. 31, 7796-7801.

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Eijsink, V. G. H., Vriend, G., Van den Burg, B., Venema, G. & Stulp, B.K. (1990). Contribution of the C-terminal amino acid to the stability ofBacillus subtilis neutral protease. Protein Engineering 4, 99-104.

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Eijsink, V. G. H., Van der Zee, J. R., Van den Burg, B., Vriend, G. &Venema, G. (1991a). Improving the thermostability of the neutralprotease of Bacillus stearothermophilus by replacing a buried asparagineby leucine. FEBS letters 282, 13-16.

Eijsink, V. G. H., Van den Burg, B. & Venema, G. (1991b). Highperformance affinity chromatography of Bacillus neutral proteases.Biotechnol. Appl. Biochem. 14, 275-283

Eijsink, V. G. H., Van den Burg, B., Vriend, G., Berendsen, H. J. C., &Venema, G. (1991c). Thermostability of Bacillus subtilis neutralprotease. Biochem. International 24, 517-525.

Eijsink, V. G. H., Vriend, G., Van der Zee, J. R., Van den Burg, B. &Venema, G. (1992a). Increasing the thermostability of the neutralprotease of Bacillus stearothermophilus by improvement of internalhydrogen bonding. Biochem. J. 285, 625-628.

Eijsink, V. G. H., Dijkstra, B. W., Vriend, G., van der Zee, J. R.,Veltman, O. R., van der Vinne, B., van den Burg, B., Kempe, S. & Venema,G. (1992b). The effect of cavity-filling mutations on thethermostability of B. stearothermophilus neutral protease Protein Engng.5,421-426.

Eijsink, V. G. H., Vriend, G., Van der Vinne, B., Hazes, B., Van denBurg, B., & Venema, G. (1992c). Effects of changing the interactionbetween subdomains on the thermostability of Bacillus neutral proteases.Proteins Struct. Funct. Genet. 14, 224-236.

Eijsink, V. G. H., Vriend, G., Hardy, F., Veltman, O. R., van der Vinne,B., van den Burg, B., Dijkstra, B. W., van der Zee, J. R. & Venema, G.(1993). Structural determinants of the thermostability ofthermolysin-like Bacillus neutral proteases. In: Stability andstabilization of enzymes (W. F. van den Tweel et all, eds.), EsevierScience Publishers, pp. 91-99.

Fersht, A. R. & Serrano, L. (1993). principles of protein stabilityderived from protein engineering experiments. Curr. Opinion Struct.Biol. 3, 75-83.

Fujii, M., Takagi, M., Imanaka, T., & Aiba, S. (1983). Molecular cloningof a thermostable neutral protease gene from Bacillus stearothermophilusin a vector plasmid and its expression in Bacillus stearothermophilusand Bacillus subtilis. J. Bacteriol. 154, 831-837.

Geisow, M. J. (1991). Stabilizing protein products: coming in from thecold. Trends in Biotechnol. 9, 149-150.

Gerhartz (ed.) (1990). Enzymes in industry. VCH Verlagsgesellschaft,Weinheim, Germany.

Hardy, F., Vriend, G., Van der Vinne, B., Venema, G., & Eijsink, V. G.H. (1993). Stabilization of Bacillus stearothemophilus neutral proteaseby introduction of prolines. FEBS lett. 317, 89-92.

Holmes, M. A., & Matthews, B. W. (1982). Structure of thermolysinrefined at 1.6 Å resolution. J. Mol. Biol. 160, 623-639.

Imanaka, T., Shibazaki, M., & Takagi, M. (1986). A new way of enhancingthe stability of proteases. Nature 324, 695-697.

Isowa, Y., Ohmori, M., Ichikawa, T., Mori, K., Nonaka, Y., Kihara, K.,Qyama, K., Satoh, H. & Nishimura, S. (1979). The thermolysin-catalyzedcondensation reactions of N-substituted aspartic and glutamic acids withphenylalanine alkyl esters. Tetrahedron letters 28, 2611-2612.

Kristjansson, J. K. (1989). Thermophilic organisms as sources ofthermostable enzymes. Trends in Biotechnol. 7, 349-353.

Kubo, M. & Imanaka, T (1988). Cloning and nucleotide sequence of thehighly thermostable neutral protease gene from Bacillusstearothermophilus. J. Gen. Microbiol. 134, 1883-1892.

Kubo, M., Mitsuda, Y., Takagi, M. & Imanaka, T. (1992). Alteration ofspecific activity and stability of thermostable neutral protease bysite-directed mutagenesis. Appl. Environm. Microbiol. 58, 3779-3783.

Marquardt, R., Hilgenfeld, R. & Keller, R. (1990). Proteasegen ausBacillus thermoproteolyticus rokko, Verfahren zu seiner Gewinnung undseine Verwendung. European patent EP 90 11 6858/0 418 625 A1.

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Nosoh, Y. & Sekiguchi, T. (1990). Protein engineering forthermostability. Trends in Biotechnol. 8, 16-20.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., USA.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1049 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - GCAACCGATG GGGCCATTTT GAATAAGTTC AACCAAATCG ACAGCCGCCA GC -            #CCGGCGGC     60                                                                 - - GGGCAGCCGG TCGCCGGCGC GTCGACGGTC GGCGTGGGCC GGGGTGTGTT GG -            #GGGATCAG    120                                                                 - - AAATATATCA ATACGACGTA TTCCTCGTAT TACGGCTACT ACTATTTGCA AG -            #ACAATACG    180                                                                 - - CGCGGCAGCG GCATTTTTAC GTATGACGGA CGAAACCGCA CCGTTTTGCC CG -            #GCAGCTTG    240                                                                 - - TGGACCGATG GCGACAACCA ATTTACCGCC AGCTATGACG CGGCGGCCGT GG -            #ACGCCCAT    300                                                                 - - TATTACGCCG GCGTCGTGTA TGATTACTAC AAAAATGTGC ACGGCCGGCT GA -            #GCTATGAC    360                                                                 - - GGCAGCAACG CCGCCATCCG TTCGACCGTC CATTATGGCC GCGGCTACAA CA -            #ACGCGTTT    420                                                                 - - TGGAACGGTT CGCAAATGGT GTACGGCGAT GGCGACGGAC AGACGTTTTT GC -            #CGTTTTTC    480                                                                 - - GGCGGCATTG ACGTCGTGGG GCATGAGTTG ACCCATGCGG TGACGGATTA TA -            #CGGCCGGG    540                                                                 - - CTTGTTTACC AAAACGAATC TGGCGCCATC AATGAAGCGA TGTCCGATAT TT -            #TCGGCACG    600                                                                 - - CTCGTGGAGT TCTACGCCAA CCGCAACCCG GACTGGGAGA TTGGCGAAGA CA -            #TTTACACG    660                                                                 - - CCTGGGGTCG CCGGCGATGC GCTCCGCTCG ATGTCCGACC CGGCGAAATA CG -            #GCGATCCG    720                                                                 - - GATCATTATT CCAAACGGTA CACCGGCACG CAAGACAACG GCGGCGTCCA TA -            #CAAACAGC    780                                                                 - - GGCATCATCA ATAAAGCGGC GTACTTGCTC AGCCAAGGCG GCGTCCATTA TG -            #GCGTGAGC    840                                                                 - - GTCAACGGCA TCGGCCGCGA CAAAATGGGG AAAATTTTCT ACCGGGCGCT TG -            #TCTACTAT    900                                                                 - - TTGACGCCGA CGTCGAACTT CAGCCAGCTG CGTGCCGCCT GCGTGCAAGC GG -            #CCGCTGAT    960                                                                 - - TTGTACGGGT CGACAAGCCA AGAAGTCAAC TCGGTGAAAC AGGCGTTCAA TG -            #CGGTTGGA   1020                                                                 - - GTGTATTAAG ACGATGAGGT CGTACGCGT         - #                  - #              1049                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1040 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - GCCGTAGACG GAAAAATTTT AAATAAATTT AACCAACTTG ACGCCGCAAA AC -             #CAGGTGAT     60                                                                 - - GTGAAGTCGA TAACAGGAAC ATCAACTGTC GGAGTGGGAA GAGGAGTACT TG -            #GTGATCAA    120                                                                 - - AAAAATATTA ATACAACCTA CTCTAGCTAC TACTATTTAC AAGATAATAC GC -            #GTGGAAAT    180                                                                 - - GGGATTTTCA CGTATGATGC GAAATACCGT ACGACATTGC CGGGAAGCTT AT -            #GGGCAGAT    240                                                                 - - GCAGATAACC AATTTTTTGC GAGCTATGAT GCTCCAGCGG TTGATGCTCA TT -            #ATTACGCT    300                                                                 - - GGTGTGACAT ATGACTACTA TAAAAATGTT CATAACCGTC TCAGTTACGA CG -            #GAAATAAT    360                                                                 - - GCAGCTATTA GATCATCCGT TCATTATAGC CAAGGCTATA ATAACGCATT TT -            #GGAACGGT    420                                                                 - - TCGCAAATGG TGTATGGCGA TGGTGATGGT CAAACATTTA TTCCACTTTC TG -            #GTGGTATT    480                                                                 - - GATGTGGTCG CTCATGAGTT AACGCATGCC GTAACCGATT ATACAGCCGG AC -            #TCATTTAT    540                                                                 - - CAAAACGAAT CTGGTGCAAT TAATGAGGCA ATATCTGATA TTTTTGGAAC GT -            #TAGTCGAA    600                                                                 - - TTTTACGCTA ACAAAAATCC AGATTGGGAA ATTGGAGAGG ATGTGTATAC AC -            #CTGGTATT    660                                                                 - - TCAGGGTATT CGCTCCGTTC GATGTCCGAT CCGGCAAAGT ATGGTGATCC AG -            #ATCACTAT    720                                                                 - - TCAAAGCGCT ATACAGGCAC GCAAGATAAT GCCGGGGTTC ATATCAATAG CG -            #GAATTATC    780                                                                 - - AACAAAGCCG CTTATTTGAT TAGCCAAGGC GGTACGCATT ACGGTGTGAG TG -            #TTGTCGGA    840                                                                 - - ATCGGACGCG ATAAATTGGG GAAAATTTTC TATCGTGCAT TAACGCAATA TT -            #TAACACCA    900                                                                 - - ACGTCCAACT TTAGCCAACT TCGTGCTGCC GCTGTTCAAT CAGCCACTGA CT -            #TGTACGGT    960                                                                 - - TCGACAAGCC AGGAAGTCGC TTCTGTGAAG CAGGCCTTTG ATGCGGTAGG GG -            #TGAAATAA   1020                                                                 - - AGTGGTATCT CATCAGTGGG            - #                  - #                     104 - #0                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 319 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - Val Ala Gly Ala Ser Thr Val Gly Val Gly Ar - #g Gly Val Leu Gly Asp      1               5   - #                10  - #                15               - - Gln Lys Tyr Ile Asn Thr Thr Tyr Ser Ser Ty - #r Tyr Gly Tyr Tyr Tyr                  20      - #            25      - #            30                   - - Leu Gln Asp Asn Thr Arg Gly Ser Gly Ile Ph - #e Thr Tyr Asp Gly Arg              35          - #        40          - #        45                       - - Asn Arg Thr Val Leu Pro Gly Ser Leu Trp Th - #r Asp Gly Asp Asn Gln          50              - #    55              - #    60                           - - Phe Thr Ala Ser Tyr Asp Ala Ala Ala Val As - #p Ala His Tyr Tyr Ala      65                  - #70                  - #75                  - #80        - - Gly Val Val Tyr Asp Tyr Tyr Lys Asn Val Hi - #s Gly Arg Leu Ser Tyr                      85  - #                90  - #                95               - - Asp Gly Ser Asn Ala Ala Ile Arg Ser Thr Va - #l His Tyr Gly Arg Gly                  100      - #           105      - #           110                  - - Tyr Asn Asn Ala Phe Trp Asn Gly Ser Gln Me - #t Val Tyr Gly Asp Gly              115          - #       120          - #       125                      - - Asp Gly Gln Thr Phe Leu Pro Phe Ser Gly Gl - #y Ile Asp Val Val Gly          130              - #   135              - #   140                          - - His Glu Leu Thr His Ala Val Thr Asp Tyr Th - #r Ala Gly Leu Val Tyr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gln Asn Glu Ser Gly Ala Ile Asn Glu Ala Me - #t Ser Asp Ile Phe        Gly                                                                                             165  - #               170  - #               175             - - Thr Leu Val Glu Phe Tyr Ala Asn Arg Asn Pr - #o Asp Trp Glu Ile Gly                  180      - #           185      - #           190                  - - Glu Asp Ile Tyr Thr Pro Gly Val Ala Gly As - #p Ala Leu Arg Ser Met              195          - #       200          - #       205                      - - Ser Asp Pro Ala Lys Tyr Gly Asp Pro Asp Hi - #s Tyr Ser Lys Arg Tyr          210              - #   215              - #   220                          - - Thr Gly Thr Gln Asp Asn Gly Gly Val His Th - #r Asn Ser Gly Ile Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asn Lys Ala Ala Tyr Leu Leu Ser Gln Gly Gl - #y Val His Tyr Gly        Val                                                                                             245  - #               250  - #               255             - - Ser Val Thr Gly Ile Gly Arg Asp Lys Met Gl - #y Lys Ile Phe Tyr Arg                  260      - #           265      - #           270                  - - Ala Leu Val Tyr Tyr Leu Thr Pro Thr Ser As - #n Phe Ser Gln Leu Arg              275          - #       280          - #       285                      - - Ala Ala Cys Val Gln Ala Ala Ala Asp Leu Ty - #r Gly Ser Thr Ser Gln          290              - #   295              - #   300                          - - Glu Val Asn Ser Val Lys Gln Ala Phe Asn Al - #a Val Gly Val Tyr          305                 3 - #10                 3 - #15                            - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 316 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - Ile Thr Gly Thr Ser Thr Val Gly Val Gly Ar - #g Gly Val Leu Gly Asp      1               5   - #                10  - #                15               - - Gln Lys Asn Ile Asn Thr Thr Tyr Ser Ser Ty - #r Tyr Tyr Leu Gln Asp                  20      - #            25      - #            30                   - - Asn Thr Arg Gly Asn Gly Ile Phe Thr Tyr As - #p Ala Lys Tyr Arg Thr              35          - #        40          - #        45                       - - Thr Leu Pro Gly Ser Leu Trp Ala Asp Ala As - #p Asn Gln Phe Phe Ala          50              - #    55              - #    60                           - - Ser Tyr Asp Ala Pro Ala Val Asp Ala His Ty - #r Tyr Ala Gly Val Thr      65                  - #70                  - #75                  - #80        - - Tyr Asp Tyr Tyr Lys Asn Val His Asn Arg Le - #u Ser Tyr Asp Gly Asn                      85  - #                90  - #                95               - - Asn Ala Ala Ile Arg Ser Ser Val His Tyr Se - #r Gln Gly Tyr Asn Asn                  100      - #           105      - #           110                  - - Ala Phe Trp Asn Gly Ser Gln Met Val Tyr Gl - #y Asp Gly Asp Gly Gln              115          - #       120          - #       125                      - - Thr Phe Ile Pro Leu Ser Gly Gly Ile Asp Va - #l Val Ala His Glu Leu          130              - #   135              - #   140                          - - Thr His Ala Val Thr Asp Tyr Thr Ala Gly Le - #u Ile Tyr Gln Asn Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Gly Ala Ile Asn Glu Ala Ile Ser Asp Il - #e Phe Gly Thr Leu        Val                                                                                             165  - #               170  - #               175             - - Glu Phe Tyr Ala Asn Lys Asn Pro Asp Trp Gl - #u Ile Gly Glu Asp Val                  180      - #           185      - #           190                  - - Tyr Thr Pro Gly Ile Ser Gly Asp Ser Leu Ar - #g Ser Met Ser Asp Pro              195          - #       200          - #       205                      - - Ala Lys Tyr Gly Asp Pro Asp His Tyr Ser Ly - #s Arg Tyr Thr Gly Thr          210              - #   215              - #   220                          - - Gln Asp Asn Ala Gly Val His Ile Asn Ser Gl - #y Ile Ile Asn Lys Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Tyr Leu Ile Ser Gln Gly Gly Thr His Ty - #r Gly Val Ser Val        Val                                                                                             245  - #               250  - #               255             - - Gly Ile Gly Arg Asp Lys Leu Gly Lys Ile Ph - #e Tyr Arg Ala Leu Thr                  260      - #           265      - #           270                  - - Gln Tyr Leu Thr Pro Thr Ser Asn Phe Ser Gl - #n Leu Arg Ala Ala Ala              275          - #       280          - #       285                      - - Val Gln Ser Ala Thr Asp Leu Tyr Gly Ser Th - #r Ser Gln Glu Val Ala          290              - #   295              - #   300                          - - Ser Val Lys Gln Ala Phe Asp Ala Val Gly Va - #l Lys                      305                 3 - #10                 3 - #15                          __________________________________________________________________________

What is claimed is:
 1. A method for producing a neutral protease havinghigher thermostability than thermolysin comprising site directedmutagenesis of the DNA coding for the neutral protease of SEQ ID NO: 3at the weak regions which correspond to residues 1-25, 59-72, 106, and189 of SEQ ID NO: 3, wherein said site-directed mutagenesis providesreplacement of three amino acid residues located at residues 59, 66, and72 or 61, 66, and 72 of SEQ ID NO: 3 in the N-terminal domain of saidprotease by the corresponding residue in thermolysin.
 2. A polypeptidehaving metallo-endopeptidase activity obtainable by a method accordingto claim 1.